Methods and Compositions for Rapid Generation of Single and Multiplexed Reporters in Cells

ABSTRACT

Methods and compositions for rapid development of reporter lines utilizing safe harbor sites in iPSCS, as well as other progenitor cells, pluripotent and multipotent stem cells and differentiated cells, and multiple Lox sites are provided.

This patent application is a divisional of U.S. application Ser. No.15/536,340, filed Jun. 15, 2017, which is the U.S. National Stage ofPCT/US2015/064202, filed Dec. 7, 2015, which claims the benefit ofpriority from U.S. Provisional Application Ser. No. 62/091,792, filedDec. 15, 2014, the contents of each of which are herein incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a method for rapid development ofreporter lines utilizing safe harbor sites in iPSCS as well as otherprogenitor cells, pluripotent or multipotent stem cell, anddifferentiated cells. A master cell line which uses a Cre-recombinaseinduced cassette exchange strategy is also provided to rapidly exchangereporter cassettes to develop new reporter lines in the same isogenicbackground at high efficiency. Vector constructs used to generate theselines, as well as the selected promoters and reporters, can bemultiplexed to provide ratio-metric measurements and quantitativeanalysis as well as monitoring lineage-specific differentiation in vitroand in vivo.

BACKGROUND

Induced pluripotent stem cells (iPSC) are rapidly becoming a mainstay ofin vitro human cell-based assays for both toxicology and drug discovery.This has been possible due to a slew of advances in the field, whichinclude techniques for high efficiency homologous recombination usingtranscription activator-like effector nuclease (TALEN), zinc fingernucleases (ZFN) or clustered regularly interspaced short palindromicrepeats (CRISPRs)/cas9 system (Mali et al. Science 2013 339:823-826;Boch et al. Science 2009 326:1509-1512; Urnov et al. Nature 2005435:646-651; and Moscou, M. J. & Bogdanove, A. J. Science 2009326:1501), and the ability to make integration-free iPSC costeffectively from normal individuals and patients with monogenic andpolygenic diseases. Combined with advances in differentiating iPSC intomultiple cell types, this allows the same signaling pathways or the samemutation to be assessed in a common allelic background. The power ofthis approach has been demonstrated by multiple groups using human cellsrather than the standard xeno models used in the past (Han et al. PLoSOne 2009 4:e7155; Matsa et al. Science translational medicine 20146:239; Peng et al. Journal of biomolecular screening 2013 18:522-533;Sinnecker et al. Journal of cardiovascular translational research 20136:31-36). Other groups have used iPSC-based models to identify patientswho might adversely respond to an approved drug therapy or discover newdrugs to treat a disease (Laustriat et al. Biochemical Societytransactions 2010 38: 1051-1057; Sinnecker et al. Pharmacology &therapeutics 2014 143: 246-252; Shtrichman et al. Current molecularmedicine 2013 13:792-805; Kumar et al. Neurotoxicology 2012 33:518-529;Ananiev et al. PloS one 2011 6:e25255).

Although these efforts clearly demonstrate the utility of usingiPSC-derived cells for screening and toxicology assays, several issueshave constrained the widespread use of such cells. Some of these issuesinclude the time periods required to differentiate iPSC into anappropriate phenotype, the purity of the differentiated cells, and theconsistency of the differentiation process. Further constraints includethe lack of isogenic lines to control for allelic variability, thedifficulty in generating reporter systems, and the time required toselect stable subclones for assays (Vojnits, K. & Bremer, S. Toxicology2010 270:10-17; Fu, X. & Xu, Y. Genome medicine 2012 4:55; Ho et al.Cell transplantation 2012 21: 801-814; Sun et al. Expert review ofcardiovascular therapy 2012 10: 943-945; and Tabar, V. & Studer, L.Nature reviews. Genetics 2014 15: 82-92).

Several groups have begun to develop techniques to address theseproblems. For example researchers have shown that ZFN, TALEN andCRISPRs/cas9 systems provide efficient gene targeting technologies andallow one to develop safe harbor or lineage specific reporter system(Wang et al. Genome research 2012 22:1316-1326; Holkers et al. Nucleicacids research 2013 41: e63; Luo et al. Stem cells translationalmedicine 2014 3:821-835; Maggio et al. Scientific reports 2014 4:5105).It has also been shown that it is possible to make GFP and luciferasereporter lines using a standardized targeting system for safe harborsites where expression is not silenced during differentiation (Luo etal. Stem cells translational medicine 2014 3:821-835).

SUMMARY OF THE INVENTION

An aspect of the present invention relates to a method for developing amaster cell line. The method comprises integrating a reporter cassetteinto a cell at a safe harbor site. The reporter cassette comprises areporter gene driven by a constitutively active promoter and multipleLox sites.

Another aspect of the present invention relates to master cell lineintegrated with a reporter cassette at a safe harbor site in the cell.The reporter cassette comprises a reporter gene driven by aconstitutively active promoter and multiple Lox sites.

Yet another aspect of the present invention relates to a method ofgenerating a new reporter line using the master cell line of thisinvention. In the method a Cre-recombinase induced cassette exchangestrategy is used to exchange the reporter cassette in the master cellline with a new reporter cassette, thereby generating a new reporterline with the same isogenic background.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A through 1E. Efficient targeting of Chr. 19 and Chr. 13 safeharbor loci. Experimental strategy of generating AAVS1-copGFP (FIG. 1A)and Chr13-copGFP (FIG. 1B) iPSC lines. Solid black triangles representthe loxP sites and triangles filled with diagonal lines represent Loxsites for RMCE. Testing primer sets for “Left” (Left arm integrationtest), “Right” (Right arm integration test) and “ORF” (WT ORF test) arealso illustrated. FIG. 10 shows validation of AAVS1-copGFP heterozyteand homozygote clones by junction PCR (upper) and sequencing (lower) ofexternal genome sequence (SEQ ID NO:22)—AAVS1 Left Arm (SEQ ID NO:23)and copGFP (SEQ ID NO:24)—AAVS1 Right Arm (SEQ ID NO:25)—external genomesequence (SEQ ID NO:26). FIG. 1D shows validation of Chr13-copGFPheterozygote clone by junction PCR (upper) and sequencing (lower) ofexternal genome sequence (SEQ ID NO:27)—CRH13 Left Arm (SEQ ID NO:28)and copGFP (SEQ ID NO:24)—CHR13 Right Arm (SEQ ID NO:29)—external genomesequence (SEQ ID NO:30). FIG. 1E shows the copGFP reporter gene inAAVS-copGFP line was not silenced while differentiated into neural stemcells (NSCs). Nestin antibody was used to label the NSCs. The scale barin FIG. 1E is 100 μm.

FIGS. 2A through 2F. Rapid exchanging of reporter cassettes in safeharbors in iPSC and progenitor cells using a master cell line strategy.FIG. 2A shows the experimental strategy of quick and efficientgeneration of a reporter line via RMCE strategy. The master line,AAVS1-copGFP, was co-transfected with the Cre expression vector and thetargeting plasmid DCXp-TagGFP carrying alox2272-DCXpromoter-TagGFP-PGK-Neo-lox511 cassette for RMCE with themaster line. Cells with successfully targeted recombination wereneomycin resistant and expressed TagGFP under the endogenous promoter ofDCX instead of the constitutive CAG promoter which is seen in the masterline. Testing primers, “swap” (indicating the successful RMCE event) and“parental” (detecting the parental gene which has no swap event) werealso illustrated. Solid black triangles represent the LoxP sites andtriangles filled with diagonal lines represent Lox sites for RMCE. AfterNeomycin selection, colonies with no copGFP signal were selected underfluorescent microscope (FIG. 2B). Because the copGFP was constitutivelyactive in the master line, the cells before swap were all greenfluorescent (left; FIG. 2B). After correct swap, the CAG promoter drivencopGFP was replaced by DCX promoter driven TagGFP whose expression isoff at the iPSC stage, and cells were no longer fluorescent (right; FIG.2B). FIG. 2C shows PCR verification of the selected non-fluorescentcolonies. No “parental” PCR products were detected in any of theselected colonies. FIG. 2D shows neuronal differentiation was induced oniPSC selected above and immunostaining showed positive co-localizationof DCX AB and TagGFP, suggesting that the TagGFP is only expressed whenthe DCX gene is turned on. RMCE strategy was also tested in theprogenitor stage (NSC; FIG. 2E). AAVS1-copGFP master line NSC wereco-transfected with Cre expressing vector and the targeting plasmidDCXp-TagGFP as described in (FIG. 2A). Before transfection, allAAVS1-copGFP NSC were green fluorescent (left; FIG. 2E). After 5 dayspost transfection, cells lost green fluorescence were detected undermicroscope (spots pointed by arrows in the right graph; FIG. 2E). FIG.2F shows PCR verification of successful swapping event happened in NSC.Only “parental” PCR band was detectable in NSC before transfection.After RMCE, both “swap” and “parental” PCR products were detected in themixed culture. Scale bar is 100 μm in FIGS. 2C and 2E.

FIGS. 3A through 3D. Generation of knock-in lines at lineage specificgenes. FIG. 3A shows the experimental strategy of generatingMAP2-Nanoluc-KI. The designed ZFNs cut at the C-term of MAP2 gene beforethe stop codon. The left and right arms of MAP2 for homologousrecombination were designed to be ˜1 kb located before and after thestop codon, respectively. Testing primer sets for “Left” (MAP2 Left armintegration test), “Right” (MAP2 Right arm integration test) and “ORF”(WT MAP2 ORF test) are also illustrated. FIG. 3B shows the experimentalstrategy of generating GFAP-Nanoluc-KI. The designed ZFNs cut after thestop codon of GFAP ORF. The left and right arms of GFAP for homologousrecombination were designed to be ˜1 kb located before and after thestop codon, respectively. Testing primer sets for “Left” (GFAP Left armintegration test), “Right” (GFAP Right arm integration test) and “ORF”(WT GFAP ORF test) are also illustrated. FIG. 3C shows validation ofMAP2-Nanoluc-KI clone by junction PCR (upper) and sequencing (lower) ofexternal genome sequence (SEQ ID NO:31)—MAP2 Left Arm with P2A (SEQ IDNO:32) and P2A Nanoluc (SEQ ID NO:33)—MAP2 Right Arm (SEQ IDNO:34)—external genome sequence (SEQ ID NO:35). FIG. 3D shows validationof GFAP-Nanoluc-KI clone by junction PCR (upper) and sequencing (lower)of external genome sequence (SEQ ID NO:36)—GFAP Left Arm with P2A (SEQID NOL37) and P2A with Nanoluc (SEQ ID NO: 33)—GFAP Right Arm (SEQ IDNO:38)—external genome sequence (SEQ ID NO:39).

FIGS. 4A through 4G: Functional validation of lineage-specificexpression. FIG. 4A as is a bar graph showing an increase of luciferaselevel in culture media detected in the GFAP-Nanoluc-KI cell lines duringdirected differentiation into astrocytes. Luciferase levels shown in thebar graph were normalized by the basal level detected at day 0 inGFAP-Nanoluc-KI NSC. Immunostaining showed excellent co-localization ofHaloTag and GFAP antibodies in the GFAP-Nanoluc-KI astrocytes (D23 postdifferentiation; FIG. 4B). Live staining of HaloTag in theGFAP-Nanoluc-KI cell line before (left) and after (right) directeddifferentiation into astrocytes as shown in FIG. 4C. As shown in FIG.4D, an increase of luciferase level in culture media was detected in theMAP2-Nanoluc-KI cell lines during directed differentiation into neurons.Luciferase levels shown in the bar graph were normalized by the basallevel detected at day 0 in the MAP2-Nanoluc-KI NSC. Immunostainingshowed excellent co-localization of HaloTag and MAP2 antibodies in theMAP2-Nanoluc-KI neurons (D18 post differentiation; FIG. 4E). Livestaining of HaloTag in the MAP2-Nanoluc-KI cell line before (left; FIG.4F) and after (right; FIG. 4F) directed differentiation into neurons. Aseries dilution of GFAP-Nanoluc-KI (left; FIG. 4G) and MAP2-Nanoluc-KI(right; FIG. 4G) iPSC were plated and luciferase level from the mediawas tested. The minimum cell amount needed was 10K for detectableluciferase level of both GFAP-Nanoluc-KI and MAP2-Nanoluc-KI iPSC lines.Scale bars shown in FIGS. 4B, 4C, 4E and 4F are all 100 μm.

FIG. 5. Summary of different approaches to generate reporter lines insafe harbors and in endogenous lineage-specific genes. Geneticmodification techniques (ZFNs or TALEN) were used in combination withcarefully designed donor vectors to target and modifygenes/loci-of-interest in selected parental iPSC lines. The parentaliPSC can be well-established control lines, patient-derived lines,pre-engineered lines or master lines for the quick swapping strategy.Depending on the donor vectors and targeting genes/loci, parental iPSCcan be engineered or re-engineered into different lines expressingeither constitutively active reporter genes at the safe harbors orreporter genes that are in frame downstream of lineage-specific genes.These targeted genetically engineered iPSC can be derived intoprogenitor cells and further differentiated into different cell typesfor numerous screening purposes. Additionally, a master line cassetteexchange strategy was developed providing the opportunity to quickly andefficiently generate different reporter lines at the safe harbor sites.Using this strategy, successful targeting to both iPSC and theprogenitor cells (solid arrows) was demonstrated. It is expected thatthis strategy can also be applied directly to the differentiated cells(dotted arrow).

SEQUENCE LISTING

The nucleic and amino acid sequences disclosed herein use standardletter abbreviations for nucleotide bases, and three letter code foramino acids, as defined in 37 C.F.R. 1.822. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.Sequence names for SEQ ID NOs 1-21 as set forth in the Sequence Listingprovided herewith are as follows:

SEQ ID NO: Sequence name 1 Upstream CLYBL target 2 Upstream CLYBL TALEbinding domain 3 Downstream CLYBL target 4 Downstream CLYBL TALE bindingdomain 5 Upstream TALEN - Includes Δ152 N-terminus and +63 C-terminus 6Downstream TALEN - Includes Δ152 N-terminus and +63 C-terminus 7Upstream CLYBL TALE binding domain 8 Upstream TALEN - Includes Δ152N-terminus and +63 C-terminus 9 pZT-C13-L 10 Downstream CLYBL TALEbinding domain 11 Downstream TALEN - Includes Δ152 N-terminus and +63C-terminus 12 pZT-C13-R 13 FokI Nuclease 14 FokI Nuclease 15 Nuclearlocalization signal 16 Nuclear localization signal 17 FLAG tag 18 FLAGtag 19 CLYBL target region 20 Primer 21 Primer

DETAILED DESCRIPTION OF THE INVENTION

Induced pluripotent stem cells (iPSC) are important tools for drugdiscovery assays and toxicology screens. The present invention providesa unique platform for rapidly developing custom single or dual reportersystems for screening assays in iPSCs, other progenitor cells,pluripotent and multipotent stem cells and differentiated cells.

Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8). In order to facilitatereview of the various embodiments of this disclosure, the followingexplanations of specific terms are provided:

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Cell Culture: Cells grown under controlled condition. A primary cellculture is a culture of cells, tissues or organs taken directly from anorganism and before the first subculture. Cells are expanded in culturewhen they are placed in a growth medium under conditions that facilitatecell growth and/or division, resulting in a larger population of thecells. When cells are expanded in culture, the rate of cellproliferation is typically measured by the amount of time required forthe cells to double in number, otherwise known as the doubling time.

Differentiation: The process whereby relatively unspecialized cells(e.g., embryonic cells or stem cells) acquire specialized structuraland/or functional features characteristic of mature cells. Similarly,“differentiate” refers to this process. Typically, duringdifferentiation, cellular structure alters and tissue-specific proteinsand properties appear.

Differentiation medium: A synthetic set of culture conditions with thenutrients necessary to support the growth or survival of microorganismsor culture cells, and which allows the differentiation of cells, such asmesenchymal stem cells.

Donor polynucleotide: A polynucleotide that is capable of specificallyinserting into a genomic locus.

Downstream: A relative position on a polynucleotide, wherein the“downstream” position is closer to the 3′ end of the polynucleotide thanthe reference point. In the instance of a double-strandedpolynucleotide, the orientation of 5′ and 3′ ends are based on the sensestrand, as opposed to the antisense strand.

Embryonic Stem (ES) Cells: Pluripotent cells isolated from the innercell mass of the developing blastocyst, or the progeny of these cells.“ES cells” can be derived from any organism. ES cells can be derivedfrom mammals, including mice, rats, rabbits, guinea pigs, goats, pigs,cows, monkeys and humans. In specific, non-limiting examples, the cellsare human or murine. Without being bound by theory, ES cells cangenerate a variety of the cells present in the body (bone, muscle, braincells, etc.), provided they are exposed to conditions conducive todeveloping these cell types. Methods for producing murine ES cells canbe found in U.S. Pat. No. 5,670,372, which is herein incorporated byreference. Methods for producing human ES cells can be found in U.S.Pat. No. 6,090,622, WO 00/70021 and WO 00/27995, which are hereinincorporated by reference.

Effective amount or Therapeutically effective amount: The amount ofagent, such a cell, for example MSCs, that is sufficient to prevent,treat, reduce and/or ameliorate the symptoms and/or underlying causes ofany disorder or disease, or the amount of an agent sufficient to producea desired effect on a cell. In one embodiment, a “therapeuticallyeffective amount” is an amount sufficient to reduce or eliminate asymptom of a disease. In another embodiment, a therapeutically effectiveamount is an amount sufficient to overcome the disease itself.

Exogenous: Not normally present in a cell, but can be introduced bygenetic, biochemical or other methods. Exogenous nucleic acids includeDNA and RNA, which can be single or double-stranded; linear, branched orcircular; and can be of any length. By contrast, an “endogenous”molecule is one that is normally present in a particular cell at aparticular developmental stage under particular environmentalconditions.

Expand: A process by which the number or amount of cells in a culture isincreased due to cell division. Similarly, the terms “expansion” or“expanded” refers to this process. The terms “proliferate,”“proliferation” or “proliferated” may be used interchangeably with thewords “expand,” “expansion” or “expanded.” Typically, during anexpansion phase, the cells do not differentiate to form mature cells.

Expansion medium: A synthetic set of culture conditions suitable for theexpansion of cells, such as mesenchymal stem cells. Tissue culture mediagenerally include a carbon source, a nitrogen source and a buffer tomaintain pH. In one embodiment, a medium contains a minimal essentialmedia, such as DMEM, supplemented with various nutrients to enhancemesenchymal stem cell growth.

Additionally, the minimal essential media may be supplemented withadditives such as horse, calf or fetal bovine serum.

FokI nuclease: A nonspecific DNA nuclease that occurs naturally inFlavobacterium okeanokoites. The term includes fragments of the FokInuclease protein that retain nuclease activity that are, or may be,fused to a DNA-binding polypeptide.

Genomic insertion site: A site of the genome that is targeted for, orhas undergone, insertion of an exogenous polynucleotide.

Growth factor: A substance that promotes cell growth, survival, and/ordifferentiation. Growth factors include molecules that function asgrowth stimulators (mitogens), molecules that function as growthinhibitors (e.g. negative growth factors) factors that stimulate cellmigration, factors that function as chemotactic agents or inhibit cellmigration or invasion of tumor cells, factors that modulatedifferentiated functions of cells, factors involved in apoptosis, orfactors that promote survival of cells without influencing growth anddifferentiation. Examples of growth factors are bFGF, epidermal growthfactor (EGF), CNTF, HGF, nerve growth factor (NGF), and actvin-A.

Heterologous: A heterologous sequence is a sequence that is not normally(i.e. in the wild-type sequence) found adjacent to a second sequence. Inone embodiment, the sequence is from a different genetic source, such asa virus or organism, than the second sequence.

Induced pluripotent stem cell” (“iPS” cell or “iPSC”): A pluripotentstem cell artificially derived from a non-pluripotent cell, typically anadult somatic cell, by recombinant expression of specific factors in thenon-pluripotent cell. Factors that may be used to for iPSCs include, butare not limited to, one or more of Oct-3/4, certain members of the Soxgene family (Sox1, Sox2, Sox3, and Sox15, Klf family members (Klf1,Klf2, Klf4, and Klf5), factors of the Myc family (c-myc, L-myc, andN-myc), Nanog, and LIN28, as defined by current knowledge in the art.Other factors or methods useful for creating iPSCs are also known in theart and are considered to produce cells that fall within the scope ofthis definition.

Isolated: An “isolated” biological component (such as a nucleic acid,peptide or cell) has been substantially separated, produced apart from,or purified away from other biological components or cells of theorganism in which the component naturally occurs, i.e., otherchromosomal and extrachromosomal DNA and RNA, cells and proteins.Nucleic acids, peptides and proteins which have been “isolated” thusinclude nucleic acids and proteins purified by standard purificationmethods. The term also embraces nucleic acids, peptides and proteinsprepared by recombinant expression in a host cell as well as chemicallysynthesized nucleic acids.

Lineage-specific: Characteristics of a cell that indicate the cell willbecome one of a limited number of related cell types or a particularcell type, such as a differentiated cell or a cell undergoing theprocess of differentiation into a specific cell type or a mature celltype.

Mesenchymal Stem Cell (MSC): Also referred to as multipotent stromalcells and meant to be inclusive not only of MSCs but also of cells withreplicative potential similar thereto that can differentiate into avariety of cell types. Additional examples of cells meant to beencompassed herein by the terms MSC and/or mesenchymal stem cellsinclude, but are not limited to, mesenchymal precursor cells or MPCs,mesenchymal progenitor cells such as described by Mesoblast, Ltd., andother adult-derived stem cells such as MULTISTEM (Athersys, Inc.). Whilethese multipotent stem cells are traditionally found in the bone marrow,they can also be isolated from other tissues including, but not limitedto, cord blood, peripheral blood, fallopian tube, fetal liver and lung,placenta and fat. MSCs and other adult stem cells which can be used inaccordance with the present invention, differentiate to form cellsand/or tissues including, but not limited, adipocytes, cartilage, bone,tendons, muscle, and skin as well as myocytes, neurons and glia.

Modulate: A change in the content of genomic DNA gene. Modulation caninclude, but is not limited to, gene activation, gene repression, genedeletion, polynucleotide insertion, and polynucleotide excision.

Neural cell: A cell that exhibits a morphology, a function, and aphenotypic characteristic similar to that of glial cells and neuronsderived from the central nervous system and/or the peripheral nervoussystem.

There are several types of neurons (neuronal cells). Cholinergic neuronsmanufacture acetylcholine. GABAergic neurons manufacture gammaaminobutyric acid (GABA). Glutamatergic neurons manufacture glutamate.Dopaminergic neurons manufacture dopamine. Serotonergic neuronsmanufacture serotonin.

Neuronal stem cell or neural stem cell (NSC): Undifferentiated,multipotent, self-renewing neural cell. A NSC is a multipotent stem cellwhich is able to divide and, under appropriate conditions, hasself-renewal capability and can terminally differentiate into neurons,astrocytes, and oligodendrocytes. Hence, the neural stem cell is“multipotent” because stem cell progeny have multiple differentiationpathways. A NSC is capable of self maintenance, meaning that with eachcell division, at least one daughter cell will also be, on average, astem cell. Neural stem cells can be derived from tissues including, butnot limited to brain and spinal cord. A “long term” NSC divides inculture for at least 15 cell divisions, such as at least 15, 20, 25, 30,35, 40, 45 or 50 cell divisions. A long term retains the properties of aneuronal stem cell, such as expression of nestin and sox1, and has thecapacity to differentiate into neurons and glia in appropriate cultureconditions in vitro.

NSCs can be obtained from a cadaver or living subject, including fromfetal tissue and adult brain biopsies. NSCs can be produced from otherstem cells, such as induced pluripotent stem cells or embryonic stemcells. NSCs can be autologous or heterologous to a recipient.

Neurological disorder: A disorder in the nervous system, including thecentral nervous system (CNS) and peripheral nervous system (PNS).Examples of neurological disorders include Parkinson's disease,Huntington's disease, Alzheimer's disease, severe seizure disordersincluding epilepsy, familial dysautonomia as well as injury or trauma tothe nervous system, such as neurotoxic injury or disorders of mood andbehavior such as addiction, schizophrenia and amyotrophic lateralsclerosis. Neuronal disorders also include Lewy body dementia, multiplesclerosis, epilepsy, cerebellar ataxia, progressive supranuclear palsy,amyotrophic lateral sclerosis, affective disorders, anxiety disorders,obsessive compulsive disorders, personality disorders, attention deficitdisorder, attention deficit hyperactivity disorder, Tourette Syndrome,Tay Sachs, Nieman Pick, and other lipid storage and genetic braindiseases and/or schizophrenia.

Neurodegenerative disorder: An abnormality in the nervous system of asubject, such as a mammal, in which neuronal integrity is threatened.Without being bound by theory, neuronal integrity can be threatened whenneuronal cells display decreased survival or when the neurons can nolonger propagate a signal. Specific, non-limiting examples of aneurodegenerative disorder are Alzheimer's disease, Pantothenate kinaseassociated neurodegeneration, Parkinson's disease, Huntington's disease(Dexter et al., Brain 114:1953-1975, 1991), HIV encephalopathy(Miszkziel et al., Magnetic Res. Imag. 15:1113-1119, 1997), andamyotrophic lateral sclerosis.

Alzheimer's disease manifests itself as pre-senile dementia. The diseaseis characterized by confusion, memory failure, disorientation,restlessness, speech disturbances, and hallucination in mammals(Medical, Nursing, and Allied Health Dictionary, 4th Ed., 1994, Editors:Anderson, Anderson, Glanze, St. Louis, Mosby).

Parkinson's disease is a slowly progressive, degenerative, neurologicdisorder characterized by resting tremor, loss of postural reflexes, andmuscle rigidity and weakness (Medical, Nursing, and Allied HealthDictionary, 4th Ed., 1994, Editors: Anderson, Anderson, Glanze, St.Louis, Mosby).

Amyotrophic lateral sclerosis is a degenerative disease of the motorneurons characterized by weakness and atrophy of the muscles of thehands, forearms and legs, spreading to involve most of the body and face(Medical, Nursing, and Allied Health Dictionary, 4th Ed., 1994, Editors:Anderson, Anderson, Glanze, St. Louis, Mosby).

Pantothenate kinase associated neurodegeneration (PKAN, also known asHallervorden-Spatz syndrome) is an autosomal recessive neurodegenerativedisorder associated with brain iron accumulation. Clinical featuresinclude extrapyramidal dysfunction, onset in childhood, and arelentlessly progressive course (Dooling et al., Arch. Neurol. 30:70-83,1974). PKAN is a clinically heterogeneous group of disorders thatincludes classical disease with onset in the first two decades,dystonia, high globus pallidus iron with a characteristic radiographicappearance (Angelini et al., J. Neurol. 239:417-425, 1992), and ofteneither pigmentary retinopathy or optic atrophy (Dooling et al., Arch.Neurol. 30:70-83, 1974; Swaiman et al., Arch. Neurol 48:1285-1293,1991).

A “neurodegenerative-related disorder” is a disorder such as speechdisorders that are associated with a neurodegenerative disorder.Specific non-limiting examples of a neurodegenerative related disordersinclude, but are not limited to, palilalia, tachylalia, echolalia, gaitdisturbance, perseverative movements, bradykinesia, spasticity,rigidity, retinopathy, optic atrophy, dysarthria, and dementia.

Nucleofection: Electroporation. Nucleofection uses a combination ofelectrical parameters, generated by a device called Nucleofector, withcell-type specific reagents. The substrate is transferred directly intothe cell nucleus and the cytoplasm.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this invention are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the fusion proteins hereindisclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Pharmaceutical agent or “drug”: A chemical compound or compositioncapable of inducing a desired therapeutic or prophylactic effect whenproperly administered to a subject or a cell. “Incubating” includes asufficient amount of time for a drug to interact with a cell.“Contacting” includes incubating a drug in solid or in liquid form witha cell.

Polynucleotide: A nucleic acid sequence (such as a linear sequence) ofany length. Therefore, a polynucleotide includes oligonucleotides, andalso gene sequences found in chromosomes. An “oligonucleotide” is aplurality of joined nucleotides joined by native phosphodiester bonds.An oligonucleotide is a polynucleotide of between 6 and 300 nucleotidesin length. An oligonucleotide analog refers to moieties that functionsimilarly to oligonucleotides but have non-naturally occurring portions.For example, oligonucleotide analogs can contain non-naturally occurringportions, such as altered sugar moieties or inter-sugar linkages, suchas a phosphorothioate oligodeoxynucleotide. Functional analogs ofnaturally occurring polynucleotides can bind to RNA or DNA, and includepeptide nucleic acid (PNA) molecules.

Polypeptide: Three or more covalently attached amino acids. The termencompasses proteins, protein fragments, and protein domains. A“DNA-binding” polypeptide is a polypeptide with the ability tospecifically bind DNA.

The term “polypeptide” is specifically intended to cover naturallyoccurring proteins, as well as those which are recombinantly orsynthetically produced. The term “functional fragments of a polypeptide”refers to all fragments of a polypeptide that retain an activity of thepolypeptide. Biologically functional fragments, for example, can vary insize from a polypeptide fragment as small as an epitope capable ofbinding an antibody molecule to a large polypeptide capable ofparticipating in the characteristic induction or programming ofphenotypic changes within a cell. An “epitope” is a region of apolypeptide capable of binding an immunoglobulin generated in responseto contact with an antigen. Thus, smaller peptides containing thebiological activity of insulin, or conservative variants of the insulin,are thus included as being of use.

The term “substantially purified polypeptide” as used herein refers to apolypeptide which is substantially free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In one embodiment, the polypeptide is at least 50%, for example at least80% free of other proteins, lipids, carbohydrates or other materialswith which it is naturally associated. In another embodiment, thepolypeptide is at least 90% free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In yet another embodiment, the polypeptide is at least 95% free of otherproteins, lipids, carbohydrates or other materials with which it isnaturally associated.

Conservative substitutions replace one amino acid with another aminoacid that is similar in size, hydrophobicity, etc. Examples ofconservative substitutions are shown below.

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Variations in the cDNA sequence that result in amino acid changes,whether conservative or not, should be minimized in order to preservethe functional and immunologic identity of the encoded protein. Theimmunologic identity of the protein may be assessed by determiningwhether it is recognized by an antibody; a variant that is recognized bysuch an antibody is immunologically conserved. Any cDNA sequence variantwill preferably introduce no more than twenty, and preferably fewer thanten amino acid substitutions into the encoded polypeptide. Variant aminoacid sequences may, for example, be 80%, 90% or even 95% or 98%identical to the native amino acid sequence.

Promoter: A promoter is an array of nucleic acid control sequences whichdirect transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription, such as, inthe case of a polymerase II type promoter, a TATA element. A promoteralso optionally includes distal enhancer or repressor elements which canbe located as much as several thousand base pairs from the start site oftranscription.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques. Similarly, arecombinant protein is one coded for by a recombinant nucleic acidmolecule.

Recombination: A process of exchange of genetic information between twopolynucleotides. “Homologous recombination (HR)” refers to thespecialized form of an exchange that takes place, for example, duringrepair of double-strand breaks in cells. Nucleotide sequence homology isutilized in recombination, for example using a “donor” molecule totemplate repair of a “target” molecule (i.e., the one that experiencedthe double-strand break), and is variously known as “non-crossover geneconversion” or “short tract gene conversion,” because it leads to thetransfer of genetic information from the donor to the target.

Safe harbor: A locus in the genome where a polynucleotide may beinserted without causing deleterious effects to the host cell. Examplesof safe harbor loci known to exist within mammalian cells may be foundwithin the AAVS1 gene, the CYBL gene, and the CCR5 gene.

Selectable marker: A gene introduced into a cell, such mammalian cellsin culture, for example a MSC, that confers a trait suitable forartificial selection from cells that do not possess the gene.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a FGF polypeptide will possess a relatively highdegree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp,CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881,1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988.Altschul, et al., Nature Genet., 6:119, 1994 presents a detailedconsideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul, et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a FGF polypeptide are typically characterizedby possession of at least about 75%, for example at least about 80%,sequence identity counted over the full length alignment with the aminoacid sequence of the factor using the NCBI Blast 2.0, gapped blastp setto default parameters. For comparisons of amino acid sequences ofgreater than about 30 amino acids, the Blast 2 sequences function isemployed using the default BLOSUM62 matrix set to default parameters,(gap existence cost of 11, and a per residue gap cost of 1). Whenaligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). Proteins with even greater similarity to thereference sequences will show increasing percentage identities whenassessed by this method, such as at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% sequence identity. Whenless than the entire sequence is being compared for sequence identity,homologs and variants will typically possess at least 80% sequenceidentity over short windows of 10-20 amino acids, and may possesssequence identities of at least 85% or at least 90% or 95% depending ontheir similarity to the reference sequence. Methods for determiningsequence identity over such short windows are available at the NCBIwebsite on the internet. One of skill in the art will appreciate thatthese sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided.

Specific binding: A sequence-specific, non-covalent interaction betweenmacromolecules (e.g., between a polypeptide and a polynucleotide). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. The term should not beconstrued to indicate that a macromolecule described as participating inspecific binding, or as being specific for another given macromolecule,cannot bind to another macromolecule, but rather that the specificnature of the interaction is significantly favored over a nonspecific orrandom binding. Such “specific binding” interactions are generallycharacterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹ or lower.

Subject: Human and non-human animals, including all vertebrates, such asmammals and non-mammals, such as non-human primates, mice, rabbits,sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. Inmany embodiments of the described methods, the subject is a human.

Synapse: Highly specialized intercellular junctions between neurons andbetween neurons and effector cells across which a nerve impulse isconducted (synaptically active). Generally, the nerve impulse isconducted by the release from one neuron (presynaptic neuron) of achemical transmitter (such as dopamine or serotonin) which diffusesacross the narrow intercellular space to the other neuron or effectorcell (post-synaptic neuron). Generally neurotransmitters mediate theireffects by interacting with specific receptors incorporated in thepost-synaptic cell. “Synaptically active” refers to cells (e.g.,differentiated neurons) which receive and transmit action potentialscharacteristic of mature neurons.

Transduced, Transformed and Transfected: A virus or vector “transduces”a cell when it transfers nucleic acid into the cell. A cell is“transformed” or “transfected” by a nucleic acid transduced into thecell when the DNA becomes stably replicated by the cell, either byincorporation of the nucleic acid into the cellular genome, or byepisomal replication.

Numerous methods of transfection are known to those skilled in the art,such as: chemical methods (e.g., calcium-phosphate transfection),physical methods (e.g., electroporation, microinjection, particlebombardment), fusion (e.g., liposomes), receptor-mediated endocytosis(e.g., DNA-protein complexes, viral envelope/capsid-DNA complexes) andby biological infection by viruses such as recombinant viruses (Wolff,J. A., ed, Gene Therapeutics, Birkhauser, Boston, USA, 1994). In thecase of infection by retroviruses, the infecting retrovirus particlesare absorbed by the target cells, resulting in reverse transcription ofthe retroviral RNA genome and integration of the resulting provirus intothe cellular DNA. Methods for the introduction of genes into cells areknown (e.g. see U.S. Pat. No. 6,110,743, herein incorporated byreference). These methods can be used to transduce a MSC or a cellproduced by the methods described herein.

Genetic modification of the target cell is an indicium of successfultransfection. “Genetically modified cells” refers to cells whosegenotypes have been altered as a result of cellular uptakes of exogenousnucleotide sequence by transfection. A reference to a transfected cellor a genetically modified cell includes both the particular cell intowhich a vector or polynucleotide is introduced and progeny of that cell.

Transgene: An exogenous gene.

Treating, Treatment, and Therapy: Any success or indicia of success inthe attenuation or amelioration of an injury, pathology or condition,including any objective or subjective parameter such as abatement,remission, diminishing of symptoms or making the condition moretolerable to the patient, slowing in the rate of degeneration ordecline, making the final point of degeneration less debilitating,improving a subject's physical or mental well-being, or prolonging thelength of survival. The treatment may be assessed by objective orsubjective parameters; including the results of a physical examination,neurological examination, or psychiatric evaluations.

Upstream: A relative position on a polynucleotide, wherein the“upstream” position is closer to the 5′ end of the polynucleotide thanthe reference point. In the instance of a double-strandedpolynucleotide, the orientation of 5′ and 3′ ends are based on the sensestrand, as opposed to the antisense strand.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in the host cell, such as anorigin of replication. A vector may also include one or more therapeuticgenes and/or selectable marker genes and other genetic elements known inthe art. A vector can transduce, transform or infect a cell, therebycausing the cell to express nucleic acids and/or proteins other thanthose native to the cell. A vector optionally includes materials to aidin achieving entry of the nucleic acid into the cell, such as a viralparticle, liposome, protein coating or the like.

Zinc finger DNA binding domain: A polypeptide domain that binds DNA in asequence-specific manner through one or more zinc fingers, which areregions of amino acid sequence within the binding domain whose structureis stabilized through coordination of a zinc ion.

Zinc finger binding domains, for example the recognition helix of a zincfinger, can be “engineered” to bind to a predetermined nucleotidesequence. Rational criteria for design of zinc finger binding domainsinclude application of substitution rules and computerized algorithmsfor processing information in a database storing information of existingZFP designs and binding data, see for example U.S. Pat. Nos. 5,789,538;5,925,523; 6,007,988; 6,013,453; 6,140,081; 6,200,759; 6,453,242; and6,534,261; and PCT Publication Nos. WO 95/19431; WO 96/06166; WO98/53057; WO 98/53058; WO 98/53059; WO 98/53060; WO 98/54311; WO00/27878; WO 01/60970; WO 01/88197; WO 02/016536; WO 02/099084 and WO03/016496.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of up to ±10% from the specified value. Unlessotherwise indicated, all numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forthused in the specification and claims are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are approximations that mayvary depending upon the desired properties sought to be obtained by thedisclosed subject matter. At the very least, and not as an attempt tolimit the application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contain certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, “A or B” is intended to include “A,”“B,” and “both A and B,” unless the context clearly indicates otherwise.It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The term “comprises”means “includes.” All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingexplanations of terms, will control. In addition, the materials,methods, and examples are illustrative only and not intended to belimiting.

Methods and Compositions for Rapid Generation of Single and MultiplexedReporters in Cells

An important feature of iPSCs is that they can be engineered in multipleways to be used for screenings, for developing therapeutic purposes, orfor investigating disease mechanisms or processes. For instance, iPSCscan be engineered to create ubiquitous reporters to develop enhancedassays, lineage-specific reporters to allow for stage-specificscreening, or pathway and organelle reporters to allow for focusedscreening.

Disclosed herein are compositions and methods for rapid generation ofsingle and multiplexed reporters in various iPSC cell lines as well asother progenitor cells including other pluripotent and multipotent stemcells and differentiated cells.

Efficient Targeting of Chr. 19 and Chr. 13 Safe Harbor Loci in MultipleLines with Multiple Constructs

High efficiency TALEN and ZFN were designed which target two safe harborsites on chromosome 13 and 19 in an iPSC line.

Recombinant polynucleotide-binding polypeptides for use in targetingthese chromosomes can occur in a variety of forms. In some embodiments,the recombinant polynucleotide-binding polypeptide is a recombinantDNA-binding polypeptide that specifically binds to a genomic targetsequence in the cell. In one embodiment the targeted genomic sequencebound by the recombinant DNA-binding polypeptide falls within thesequence of SEQ ID NO: 19, or its corresponding antisense sequence. Inanother embodiment the targeted sequence bound by the recombinantDNA-binding polypeptide in the genome of the cell includes the sequenceof SEQ ID NO: 1. In yet another embodiment, the targeted sequence boundby the recombinant DNA-binding polypeptide is the sequence of SEQ IDNO: 1. Alternatively, the targeted sequence bound by the recombinantDNA-binding polypeptide may include a sequence that is antisense, orcomplementary, to the sequence of SEQ ID NO: 1. In one embodiment, thetargeted sequence bound by the recombinant DNA-binding polypeptide is asequence that is antisense, or complementary, to the sequence of SEQ IDNO: 1. In another embodiment the targeted sequence bound by therecombinant DNA-binding polypeptide includes the sequence of SEQ ID NO:3. In a further embodiment, the targeted sequence bound by therecombinant DNA-binding polypeptide is the sequence of SEQ ID NO: 3.Alternatively, the targeted sequence bound by the recombinantDNA-binding polypeptide can include a sequence that is antisense, orcomplementary, to the sequence of SEQ ID NO: 3. In one embodiment, thetargeted sequence bound by the recombinant DNA-binding polypeptide is asequence that is antisense, or complementary, to the sequence of SEQ IDNO: 3.

In some embodiments the described recombinant DNA-binding polypeptideincludes a zinc-finger domain or a transcription activator-like effector(TALE) domain, or a polypeptide fragment thereof that retains the DNAbinding function of the TALE domain or the zinc-finger domain.Furthermore, the recombinant DNA-binding polypeptide may also becombined with a polypeptide having nuclease activity, such as azinc-finger domain or a transcription activator-like effector (TALE)domain fused to a nuclease protein, or a fragment thereof. Exemplarynucleases include, but are not limited to, S1 nuclease, mung beannuclease, pancreatic DNAase I, micrococcal nuclease, and yeast HOendonuclease (see also Linn et al. (eds.) Nucleases, Cold Spring HarborLaboratory Press, 1993).

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme Fok I catalyzesdouble-stranded cleavage of DNA, at nine nucleotides from itsrecognition site on one strand and 13 nucleotides from its recognitionsite on the other (see, for example, U.S. Pat. Nos. 5,356,802; 5,436,150and 5,487,994; Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279;Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al.(1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J.Biol. Chem. 269:31, 978-31, 982). Thus, in one embodiment, a nucleasedomain from at least one Type IIS restriction enzyme is utilized. Anexemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is FokI. This particular enzyme isactive as a dimer. See Bitinaite et al. (1998) Proc. Natl. Acad. Sci.USA 95: 10,570-10,575. Additional forms of FokI nuclease are set forthin U.S. Published Patent Application No. 20110027235, which isincorporated herein by reference.

In some embodiments the polypeptide having nuclease activity that isfused with the recombinant DNA-binding polypeptide is the FokI nuclease,or a derivative or fragment thereof that retains the nuclease activity.In some embodiments, the FokI nuclease is at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or about100% identical to SEQ ID NO: 13.

In the case of a recombinant DNA-binding polypeptide produced from aTALE domain, fusion with a polypeptide having nuclease activity forms atranscription activator-like effector nuclease (TALEN). Some of theTALEN embodiments described herein are designed to specifically target agenomic sequence that falls within the sequence of SEQ ID NO: 19, or itscorresponding antisense sequence, such as, for example, the sequence ofSEQ ID NO: 1 or 3. In one embodiment the targeted sequence bound by adescribed TALE domain includes the sequence of SEQ ID NO: 1. In oneembodiment, the targeted sequence bound by a described TALE domain isthe sequence of SEQ ID NO: 1. Alternatively, the targeted sequence boundby a described TALE domain may include a sequence that is antisense, orcomplementary, to the sequence of SEQ ID NO: 1. In one embodiment, thetargeted sequence bound by a described TALE domain is a sequence that isantisense, or complementary, to the sequence of SEQ ID NO: 1. In anotherembodiment the targeted sequence bound by a described TALE domainincludes the sequence of SEQ ID NO: 3. In one embodiment, the targetedsequence bound by a described TALE domain is the sequence of SEQ ID NO:3. Alternatively, the targeted sequence bound by a described TALE domainmay include a sequence that is antisense, or complementary, to thesequence of SEQ ID NO: 3. In one embodiment, the targeted sequence boundby a described TALE domain is a sequence that is antisense, orcomplementary, to the sequence of SEQ ID NO: 3.

The TALE domains of use in the methods disclosed herein can be linked toa polypeptide having nuclease activity to form a TALEN, which can beused to cleave DNA at a specific location of interest. In one embodimentthe targeted sequence bound by a described TALEN includes the sequenceof SEQ ID NO: 1. In one embodiment, the targeted sequence bound by adescribed TALEN is the sequence of SEQ ID NO: 1. Alternatively, thetargeted sequence bound by a described TALEN may include a sequence thatis antisense, or complementary, to the sequence of SEQ ID NO: 1. In oneembodiment, the targeted sequence bound by a described TALEN is asequence that is antisense, or complementary, to the sequence of SEQ IDNO: 1. In another embodiment the targeted sequence bound by a describedTALEN includes the sequence of SEQ ID NO: 3. In one embodiment, thetargeted sequence bound by a described TALEN is the sequence of SEQ IDNO: 3. Alternatively, the targeted sequence bound by a described TALENmay include a sequence that is antisense, or complementary, to thesequence of SEQ ID NO: 3. In one embodiment, the targeted sequence boundby a described TALEN is a sequence that is antisense, or complementary,to the sequence of SEQ ID NO: 3.

For the methods disclosed herein, the recombinant DNA-bindingpolypeptide may also be combined with a polypeptide having nucleaseactivity, such as a zinc-finger domain or a transcription activator-likeeffector (TALE) domain fused to a nuclease protein, or a fragmentthereof. In some embodiments the polypeptide having nuclease activitythat is fused with the recombinant DNA-binding polypeptide is the fokInuclease, or a derivative or fragment thereof that retains the nucleaseactivity. In the case of a recombinant DNA-binding polypeptide producedfrom a TALE domain, fusion with a polypeptide having nuclease activityforms a transcription activator-like effector nuclease (TALEN).

Some of the TALEN embodiments of use in the disclosed methods aredesigned to specifically target a genomic sequence that falls within thesequence of SEQ ID NO: 19, or its corresponding antisense sequence, suchas, for example, the sequence of SEQ ID NO: 1 or 3. In one embodimentthe TALE domain includes the amino acid sequence of SEQ ID NO: 7. Inanother embodiment the TALE domain includes an amino acid sequence ofSEQ ID NO: 10. In further embodiments a TALE domain is fused to apolypeptide having nuclease activity to form a TALEN. One TALEN of usein the methods disclosed herein is a TALE domain that includes the aminoacid sequence of SEQ ID NO: 7 incorporated into a polypeptide havingnuclease activity. In one such embodiment, the amino acid sequence ofSEQ ID NO: 7 is incorporated into a polypeptide that also includes afokI nuclease, or a fragment thereof. For example, the amino acidsequence of SEQ ID NO: 7 may be incorporated into a polypeptide thatalso includes the amino acid sequence of SEQ ID NO: 13. One embodimentof a polypeptide where the amino acid sequence of SEQ ID NO: 7 isincorporated with the amino acid sequence of SEQ ID NO: 13, is thepolypeptide of SEQ ID NO: 8. One TALEN of use in the methods disclosedherein is a TALE domain that includes the amino acid sequence of SEQ IDNO: 10 incorporated into a polypeptide having nuclease activity. In onesuch embodiment, the amino acid sequence of SEQ ID NO: 10 isincorporated into a polypeptide that also includes a fokI nuclease, or afragment thereof that retains nuclease activity. For example, the aminoacid sequence of SEQ ID NO: 10 may be incorporated into a polypeptidethat also includes the amino acid sequence of SEQ ID NO: 13. Oneembodiment of a polypeptide where the amino acid sequence of SEQ ID NO:10 is incorporated with the amino acid sequence of SEQ ID NO: 13, is thepolypeptide of SEQ ID NO: 11.

The TALE constructs of use in the methods disclosed herein can be usedto target specific DNA sequences, such as a genomic sequence of interestin an MSC. When coupled with a polypeptide having nuclease activity toform a TALEN, these constructs can be used to target a specificpolynucleotide of interest for modification in the genome of the MSC. Inone embodiment the described TALE domain includes the amino acidsequence of SEQ ID NO: 7 which can target the sequence of SEQ ID NO: 1specifically. In another embodiment the TALE domain includes an aminoacid sequence of SEQ ID NO: 10 which can target the sequence of SEQ IDNO: 3 specifically. In further embodiments a described TALE domain isfused to a polypeptide having nuclease activity to form a TALEN. OneTALEN described herein is a TALE domain that includes the amino acidsequence of SEQ ID NO: 7 incorporated into a polypeptide having nucleaseactivity, which can target the sequence of SEQ ID NO: 1 specifically. Inone such embodiment, the amino acid sequence of SEQ ID NO: 7 isincorporated into a polypeptide that also includes a fokI nuclease, or afragment thereof that retains nuclease activity, and can target thesequence of SEQ ID NO: 1 specifically and mediate cleavage of a DNAsequence proximal to the segment where the polynucleotide is bound. Forexample, the amino acid sequence of SEQ ID NO: 7 may be incorporatedinto a polypeptide that also includes the amino acid sequence of SEQ IDNO: 13, for specific targeting of the sequence of SEQ ID NO: 1 andcleavage of the polynucleotide sequence proximal to the binding locus.One embodiment of a polypeptide where the amino acid sequence of SEQ IDNO: 7 is incorporated with the amino acid sequence of SEQ ID NO: 13, isthe polypeptide of SEQ ID NO: 8, which can specifically bind thesequence of SEQ ID NO: 1 and cleave the polynucleotide sequence proximalto the binding locus.

Another TALEN of use in the methods disclosed herein is a TALE domainthat includes the amino acid sequence of SEQ ID NO: 10 incorporated intoa polypeptide having nuclease activity, which can target the sequence ofSEQ ID NO: 3 specifically. In one such embodiment, the amino acidsequence of SEQ ID NO: 10 is incorporated into a polypeptide that alsoincludes a fokI nuclease, or a fragment thereof that retains nucleaseactivity, and can target the sequence of SEQ ID NO: 3 specifically andmediate cleavage of a DNA sequence proximal to the segment where thepolynucleotide is bound. For example, the amino acid sequence of SEQ IDNO: 10 may be incorporated into a polypeptide that also includes theamino acid sequence of SEQ ID NO: 13, for specific targeting of thesequence of SEQ ID NO: 3 and cleavage of the polynucleotide sequenceproximal to the binding locus. One embodiment of a polypeptide where theamino acid sequence of SEQ ID NO: 10 is incorporated with the amino acidsequence of SEQ ID NO: 13, is the polypeptide of SEQ ID NO: 11, whichcan specifically bind the sequence of SEQ ID NO: 3 and cleave thepolynucleotide sequence proximal to the binding locus.

Modifications can be made to the described subject matter resulting insubstantially similar polypeptides and constructs that carry outessentially the same functions, in substantially the same way, as thedescribed polynucleotide-binding polypeptides and related nucleaseconstructs. For example, zinc-finger-based constructs, or CRISPRtechnology, can be used to target the loci described herein to modify agenome of a cell or chromosomal DNA. Accordingly, such variations areconsidered to be within the scope of the present disclosure.

Polynucleotides and vectors are also of use in the methods disclosedherein. The polynucleotides encode the polypeptides disclosed above. Insome embodiments, the polynucleotides and vectors encode recombinantDNA-binding polypeptides, zinc-finger or TALE domains, nuclease proteinsor polypeptides, fusion proteins produced from the fusion of DNA-bindingpolypeptides and nuclease proteins or polypeptides, such as TALENs. Insome embodiments the expression of the polypeptides encoded by thevectors are controlled by an inducible promoter. Suitable promotersinclude, but are not limited to, the doubecourtin (DCX) promoter andglial fibrillary acidic protein (GFAP). In other embodiments theexpression of the polypeptides encoded by the vectors are controlled bya repressible promoter. Cells of the present invention can be modifiedby the described vectors, for example transfected cells or cells havingan expression product of the vectors.

The polypeptides described herein can be encoded by a variety ofpolynucleotides due to the degeneracy of the genetic code. Thus, thepolynucleotides provided herein may be altered to encode the samecorresponding amino acid sequences disclosed herein, as would beunderstood by those skilled in the art. Accordingly, the use of suchvaried polynucleotide sequences should be considered within the scope ofthe presently claimed methods. The amino acid sequence of SEQ ID NO: 7may be encoded by a nucleotide having the sequence of SEQ ID NO: 2. Theamino acid sequence of SEQ ID NO: 8 may be encoded by a nucleotidehaving the sequence of SEQ ID NO: 5. The amino acid sequence of SEQ IDNO: 10 may be encoded by a nucleotide having the sequence of SEQ ID NO:4. The amino acid sequence of SEQ ID NO: 11 may be encoded by anucleotide having the sequence of SEQ ID NO: 6. The amino acid sequenceof SEQ ID NO: 13 may be encoded by a nucleotide having the sequence ofSEQ ID NO: 14.

Furthermore, the vectors of use in the methods disclosed herein, thatexpress the polynucleotides, or produce the polypeptides, may besubstituted for other vectors having similar functional capabilitiesthat would be understood by those skilled in the art having benefit ofthe present disclosure. In one embodiment, the polypeptide of SEQ ID NO:8 may be produced by the polynucleotide of SEQ ID NO: 9. In anotherembodiment the polypeptide of SEQ ID NO: 11 may be encoded by thepolynucleotide of SEQ ID NO: 12. Provided herein are donorpolynucleotides that may be inserted into the genome of the cell. Insome embodiments the donor polynucleotides are double-strandedpolynucleotides with sense and/or antisense strand polynucleotideoverhangs that are at least partially complementary to correspondingpolynucleotide overhangs of cleaved genomic DNA to facilitate insertionof the donor polynucleotide with the cleaved genomic DNA. In additionalembodiments the donor polynucleotides are single-strandedpolynucleotides with sense and/or antisense strand polynucleotideoverhangs (portions) that are at least partially complementary tocorresponding polynucleotide overhangs of cleaved genomic DNA tofacilitate insertion of the donor polynucleotide with the cleavedgenomic DNA. In some embodiments the donor polynucleotide may express apolypeptide once inserted into the genome of the cell or a celldifferentiated therefrom. In some embodiments the expressed polypeptidecan be a protein that can function to induce cell differentiation ormaturation to proceed in a particular manner, such as toward a specificcell lineage. In some embodiments the expression of a polypeptide by thedonor polynucleotide may be controlled by an inducible promoter, such asa promoter expressed in differentiated cells. In other embodiments, theexpression of a polypeptide by the donor polynucleotide may becontrolled by a repressible promoter. In still other embodiments thedonor polynucleotide may encode more than one polypeptide, for example,the donor polynucleotide may include an expression cassette having aplurality of genes. In certain embodiments where the donorpolynucleotide encodes more than one polypeptide, the donorpolynucleotide may have inducible promoters to regulate the expressionof certain genes and repressible promoters to regulate the expression ofother genes.

As shown herein, these sites can be targeted in multiple iPSC lines togenerate reporter systems while retaining pluripotent characteristics.These sites have previously been shown not to be silenced (Luo et al.Stem cells translational medicine 2014 3:821-835; Macarthur et al. Stemcells and development 2012 21:191-205). Different promoters wereevaluated and the CMV early enhancer/chicken beta actin (CAG) promoterappeared the most stable and was used for subsequent experiments.Additionally, two different reporters were evaluated: Nanoluc(luciferase) for quantitation and sensitivity, and copGFP for itsfluorescence intensity and stability. Both reporters worked efficientlyand a subset of the data is shown in FIG. 1.

The constructs and the schemas of generating knock in (KI) iPSC lines atthe two safe harbor sites with the exemplary reporter copGFP driven bythe constitutively active CAG promoter are illustrated in FIG. 1A-B. Awell-characterized integration-free iPSC line, XCL1, was used as theparental line for all gene-targeting work described herein unlessspecified otherwise. The Chr 19 site was first targeted and 37 colonieswere analyzed for AAVS1-copGFP line by PCR after drug selection andsingle cell colony cloning. Of these colonies, 12 clones were targetedon one allele and 25 were targeted to both alleles. Similar targetingefficacy was observed for the Chr 13 site. A representative example of amonoallelic (heterozygote) and biallelic (homozygote) is shown in FIG.1C-D (homozygotes for the AAVS1-copGFP line and heterozygotes for theChr13-copGFP line). Further sequencing of the PCR products confirmed thesuccessful integration of donor constructs into appropriate genome loci(FIGS. 1C and 1D).

The reporter lines engineered by each of the safe harbor integrationstrategies were then validated. Genomic stability of a representativeline, the AAVS1-copGFP line was determined. When directly differentiatedtoward the neural lineage, the copGFP reporter in this KI line was notsilenced as evidenced by continuous expression of GFP in nestin-positiveneural stem cell (NSC) (FIG. 1E). No gene silencing was observed duringrandom differentiation via embryoid body formation as cells of the threegerm layers differentiated from the AAVS1-copGFP line remainedGFP-positive.

To confirm that this safe harbor KI approach can be generalized, similarreporters were created in another well-characterized integration-freeline, XCL5 (NCRM5). As an example, a Chr13-Nanoluc-halotag line wasgenerated, similar to the Chr13-copGFP line in which a Nanoluc/HaloTagreporter was used instead of copGFP. This line was differentiated to apure population of neurons or astrocytes via directed differentiation.Further, no gene silencing in these lineages was confirmed. Takentogether, these experiments demonstrate targeting at the safe harborloci to be both reliable and efficient. The cell lines obtained werestable, karyotypically normal and the reporters did not silence onrandom or directed differentiations. In addition, both sites could betargeted simultaneously, and both monoallelic and biallelic subclonescould be identified.

Rapid Exchanging of Reporter Cassettes in Safe Harbors in iPSC andProgenitor Cells Using a Master Cell Line Strategy

While ZFN and TALEN increased targeting efficiency several-fold comparedto the traditional gene targeting methods, their efficiency may not behigh enough to target non-pluripotent cells. This may be important whenthe differentiation process is very long or genes have toxic effects atsome stages. Accordingly, in the present invention, the safe harbor sitetargeting strategy was modified by utilizing constructs with multipleLox sites, which allowed for easy replacement of one reporter orpromoter with another by Cre-recombinase mediated cassette exchange(RMCE). An nonlimiting example of such a vector design is illustrated inFIG. 2A. In this construct, the CAG promoter driving the copGFP reportercassette was inserted between lox2272 and lox511 sites with theappropriate orientation for RMCE. In addition, a puromycin resistantgene flanked by two different loxP sites was inserted at the endogenouspromoter of AAVS1. Two insulator sites were also added in this line toprevent copGFP silencing. To generate new reporter lines, daughterconstructs containing any gene-specific or ubiquitous promoter driving areporter gene can be inserted between a lox2272 and a lox511 site, anddrug selection and loss of the previous insert can be used to identifyappropriate clones.

This strategy was tested by replacing GFP driven by the ubiquitous CAGpromoter in the AAVS1-copGFP line with a promoter-reporter constructusing the neuronal lineage-specific promoter doublecortin (DCX) drivingTagGFP (see FIG. 2) or Nanoluc. In the DCX daughter construct,DCXp-TagGFP, a DCX promoter driving TagGFP together with a PGK promoterdriving Neomycin resistant gene was cloned between lox2272 and lox511sites (see FIG. 2A). In order to induce the RMCE, DCXp-TagGFP constructwas co-transfected with a plasmid expressing Cre recombinase by the PGKpromoter, into an established AAVS1-copGFP (a homozygote clone) iPSCline. After ROME, colonies that had lost green fluorescence wereidentified and picked for PCR verifications. Using primers designedspecifically for targeting the “parental” and “swap” sequences, iPSCclones were identified where cassette exchange had been successful (seeFIG. 2B). Before cassette exchange, the master iPSC line AAVS1-copGFPconstitutively expresses green fluorescence and is puromycin resistant.In the presence of Cre recombinase and DCX daughter construct, thepuromycin gene was deleted via Cre-loxP mediated recombination, and“CAGp-copGFP” was replaced by the “DCXp-TagGFP-PGKp-Neo” cassette. Thus,the new reporter line, referred to herein as DCXp-TagGFP, is notpuromycin but neomycin resistant and is not fluorescent at the iPSCstage (see FIG. 2C).

To confirm functionally appropriate expression in the DCXp-TagGFPreporter line created by cassette exchange, a directed differentiationprotocol was used to induce neuronal differentiation in accordance withprocedures described by Yan et al. (Stem cells translational medicine2013 2:862-870). As the cells differentiated toward the neuronallineage, GFP-positive cells appeared (see FIG. 2D). ICC staining 6 daysafter differentiation confirmed that all green cells were specificallylocated with DCX antibody positive neurons (see FIG. 2D), validating thespecificity of the DCXp-TagGFP reporter line.

To further confirm the utility of the master iPSC line strategy, it wasdetermined whether the RMCE can be extended to intermediate/progenitorstage cells as well. For these experiments, NSC were derived from theAAVS1-copGFP iPSC line, which maintained strong green fluorescencethrough differentiation (see FIGS. 1E and 2E). Following the same RMCEprocedures as described for the iPSC (see FIG. 2A), DCXp-TagGFP daughterconstruct and Cre-expressing plasmid were co-transfected intoAAVS1-copGFP NSC. No drug selection was used to enrich cells withsuccessful RMCE, since the goal for this experiment was not to isolatesingle clones of NSC with correct cassette exchange. Instead, the entirecell population was analyzed and cells losing green fluorescence posttransfection were identified by fluorescence microscopy, indicating thesuccessful event of RMCE (see FIG. 2E). Junction PCR was used to confirmthat cassette exchange was indeed correctly induced in a subset of theNSC (see FIG. 2F). Overall these results showed that isogenic subclonescan be rapidly generated at multiple stages of differentiation, whichshould allow expression of deleterious genes at specific stages ofdevelopment.

Generation and Lineage-Specific Expression of KI Reporters

Although lineage-specific constructs for some genes are available andsome fragments are sufficiently small that they could be targeted to thesafe harbor loci (see above DCX-copGFP reporter), a KI strategy in genesthat are expressed in specific lineages is desirable as it allows forthe development of assays to identify regulators of development. ANanoluc-HaloTag construct (see FIG. 3) was selected to knock into theendogenous MAP2 locus, and the same reporter construct in endogenousGFAP allele. Monallelic lines were produced and the 3′ prime end of thegene was targeted to allow expression of normal levels of the endogenousgene. Both KI reporter lines were made in the XCL1 iPSC line to showthat isogenic subclones could be obtained. Further, the same constructhas been used in other NCRM lines. Specifically, ZFN pairs targeting theC-term of GFAP or MAP2 genes were designed and optimized. One paircutting at ˜130 bp after the stop codon of GFAP ORF and ZFNs targeting˜90 bp before the stop codon of MAP2 gene were selected for theseexperiments (FIGS. 3A and 3B). A donor vector consisting of a reportercassette of a P2A peptide, a Nanoluc luciferase gene fused with aHaloTag, followed by a neomycin resistance gene was designed to be inframe with the C-terminal of the targeted genes (see FIGS. 3A and 3B).After co-transfection with the donor vector and mRNA of the respectiveZFN pair, and appropriate drug selection, 35 colonies from each linewere picked for further analysis. Successful insertion of the reportergenes to either GFAP or MAP2 gene was confirmed by both PCR andsequencing analyses for 33 GFAP and 4 MAP2 clones (see FIGS. 3C and 3D).Four clones of each reporter line were selected and verified to beheterozygotes (see FIGS. 3C and 3D). One of each validatedGFAP-Nanoluc-KI and MAP2-Nanoluc-KI clone was chosen for furtheranalysis as described below.

The positive expression of pluripotency markers and a normal karyotypein prolonged culture of both GFAP-Nanoluc-KI and MAP2-Nanoluc-KI iPSClines were first confirmed. Next neural differentiation was induced viaNSC formation from these two iPSC lines and the expression of thereporter genes, Nanoluc and HaloTag, was tracked during lineage-specificdifferentiation (see FIG. 4). No luciferase signal was detected inGFAP-Nanoluc-KI or MAP2-Nanoluc-KI lines or NSC derived from them (seeFIGS. 4A, 4C, 4D and 4F).

For GFAP-Nanoluc-KI NSC, the expression of luciferase and HaloTag duringastrocyte differentiation was monitored using a well-establishedprotocol (Shaltouki et al. Stem cells 2013 31:941-952). Starting fromday 18 after the NSC stage, the luminescence intensity increasedgradually as the cells differentiated to astrocytes (see FIG. 4A). Inorder to visualize expression of the reporter gene duringdifferentiation, ligand that covalently binds to HaloTag was used tolabel live GFAP-Nanoluc-KI cells at different time points duringastrocyte differentiation. HaloTag-labeled fluorescent cells were onlyobserved after the differentiation, further confirming that thereporters were turned on specifically by the GFAP promoter as the cellsdifferentiated into astrocytes (see FIG. 4C). The differentiatedGFAP-Nanoluc-KI cells (D23 post differentiation) were then tested byimmunostaining and co-localization of GFAP and HaloTag antibodies wasfound in nearly 100% of the cells, indicating that the reporter genes inGFAP-Nanoluc-KI only turned on in the GFAP-positive cells (see FIG. 4B).

Using a similar strategy, the expression of Nanoluc and HaloTag reportergenes was monitored in the MAP2-Nanoluc-KI cells during neuronaldifferentiation. A 2-week differentiation protocol was used to generatea pure population of mixed neurons from the NSC (Swistowska et al. Stemcells and development 2010 19:71-82). No detectable luminescence wasobserved until 12 days post differentiation (see FIG. 4D). Nofluorescence was detected from HaloTag expression in MAP2 NSC prior todifferentiation (see FIG. 4F). Expression of HaloTag was observed asmore and more cells differentiated into neurons (see FIG. 4E).Importantly, HaloTag antibody only stained the MAP2-positive neurons,indicating the specific expression of the reporter from the endogenousMAP2 promoter. These results signify that a single luciferase or HaloTaggene is sufficiently sensitive to allow for live tracking ofdifferentiation events.

To determine the minimal numbers of cells required for detectable levelof Nanoluc, the luminescence level of both GFAP-Nanoluc-KI astrocytesand MAP2-Nanoluc-KI neurons was measured at different cell densities(see FIG. 4G). Less than 1×10⁴ cells were required for eitherGFAP-Nanoluc-KI astrocytes or MAP2-Nanoluc-KI neurons to be detected byluminescence in a 96-well format. This result suggested that theGFAP-Nanoluc-KI and MAP2-Nanoluc-KI reporters can be used to effectivelyand accurately track astrocytes or neurons during differentiation withsmall numbers of cells and in a high-throughput manner.

To further demonstrate that clones with multiple reporters can be maderapidly, a stock of NSC was generated from the MAP2-Nanoluc-KI subclonetargeting the safe harbor locus at the NSC stage. The targeted clone(the MAP2-Nanoluc-KI line) could be re-targeted and a dual reporter linecould be readily generated. Even higher efficiency was obtained when theclone was targeted at the iPSC stage, which was comparable to those seenin an untargeted line.

Thus, as shown herein, by combining safe harbor gene editing, cassetteexchange tools, and the identification of lineage specific gene loci,the present invention provides a useful means for rapidly developingsingle and multiplexed reporters that provide investigators the abilityto develop a repertoire of assays using reporters appropriate for theirparticular need. Further, the Nanoluc-HaloTag reporter constructdisclosed herein offers several advantages. For example, it allows forsimultaneous quantitative assessment and fluorescent imaging on demandfor time-lapse imaging. By using small molecule ligands to HaloTag, onealso has the advantage of choosing fluorescent signals, as its labelingis transient, which allows for other imaging modalities to be used whennecessary. In addition, antibodies to the HaloTag are available allowingantibody labeling in fixed cells for archival purposes.

Further, the limited availability of human cells of the central nervoussystem make iPSC derived neural derivatives a promising cell source fordrug discovery and for improvement of existing drug developmentworkflow, specifically for the evaluation of toxicity and efficacy oflead compounds. Most neural differentiation protocols currentlyavailable, however, produce a heterogeneous population of neuron andglial cells, making it difficult to interpret the mechanism of action ofa given compound. Using the neural lineage-specific KI reporter approachof the present invention mitigates this problem through use of a donorvector containing dual reporters of luciferase and HaloTag attached tothe C-terminal of an endogenous lineage-specific gene. For example,using this approach, the MAP2 gene was targeted for neuron-specificreporter and the GFAP gene was targeted for astrocyte-specific reporter.Lineage-specific expression of the reporters was then validated duringlineage-specific differentiation. This MAP2-Nanoluc-KI line allows forreal-time monitoring of neuronal differentiation, and the luciferaseactivity in the culture reflects the percentage of neurons. Thenon-disruptive assay format enables accurate and quantitativemeasurement of any compound on neurons specifically. Likewise, theGFAP-Nanoluc-KI line allows for tracking and quantitative measurement ofastrocytes in culture. The fact that as few as 10⁴ cells (neurons orastrocytes) from these KI lines were needed to detect the luciferaseactivity in culture media makes these reporters/assays applicable forhigh-throughput and high content screening.

Further, because drug selection is incorporated via a T2A sitedownstream of the lineage specific promoter in the present invention, itis possible to purify neurons and astrocytes for assays. Likewise, cellscan be sorted using the fluorescent label allowing one to combinescreening with gene expression analysis.

In addition, cassette exchange in iPSC and NSCs in accordance with thepresent invention showed that clones can be rapidly generated inmultiple stages of development. While experiments described in detailherein were performed in the neuronal lineage, the platform of thepresent invention has also been demonstrated to work in mesenchymal stemcells and astrocyte precursors and is expected to work with any otherintermediate progenitor as well. The ability to use multiple reportersin the same site for different purposes is an invaluable benefit tohaving to generate new reporters or new lines and test their specificityand quality each time.

An additional advantage of the present invention is that the same safeharbors, master and control lines can be used for other lineages. Thiswill allow for development of a database of drug responses and effectsof a mutation in a single pathway in multiple cell types from a singleallelic background.

The following nonlimiting examples are provided to further illustratethe present invention.

EXAMPLES Example 1: iPSC Culture and Gene Targeting by ZFN/TALENs

A subclone of each NCRM1 and NCRM5 integration-free iPSC line (NIH CRM),named XCL1 and XCL5, was obtained from XCell Science (Novato, Calif.)and used as the parental cells for all engineered work described inthese examples. iPSC were cultured as in accordance with proceduresdescribed by Lie et al. (Methods in molecular biology 2012 873: 237-246)and Zou et al. Blood 2011 117:5561-5572) and maintained in feeder-freeconditions on Matrigel (BD Biosciences, CA) coated dishes using mTeSR™1media (STEMCELL Technologies Inc., Vancouver, Canada) following themanufacturer's protocols. TALEN expression plasmids targeting safeharbor sites in Chr.13 and Chr.19 (AAVS1) were provided by NIH andcomprise sequences set forth herein in the Section entitled ZFNexpression plasmids targeting the C-term of MAP2 and GFAP genes werepurchased from Sigma (St. Louis, Mo.). Each plasmid DNA was linearizedby XbaI for mRNA production and purification following modifiedmanufacturer's protocols.

Example 2: Donor Vector Design and Construction

A backbone vector containing a puromycin resistant gene flanked by twoloxP sites and a CAG promoter driving copGFP cassette was constructedbetween the lox2272 and lox511 sites. Insulator expressing genes wereused to generate AAVS1-copGFP donor vector targeting to the AAVS1 siteat Chr.19 (see FIG. 1A). A 754 bp left homologous arm and an 838 bpright homologous arm were amplified by PCR from XCL1 (Xcell Inc, CA)gDNA and cloned into the backbone vector. For Chr13-copGFP, a similarbackbone vector was used (the puromycin resistant gene was replaced by aPGK promoter driven neomycin resistant gene) and inserted with a 832 bpleft homologous arm and a 796 bp right homologous arm amplified from theChr13 safe harbor region where designed TALENs are targeting to.

Another backbone vector containing a P2A peptide, Nanoluc reporter genefused with a downstream HaloTag, a T2A peptide in frame with a Neomycinresistant gene and a puromycin resistant gene flanked by two loxP siteswas designed and constructed for targeting different genes to generatelineage-specific reporter donor vectors. A 1069 bp left homologous armright before the stop codon of MAP2 gene was PCRed from XCL1, and thencloned into the backbone vector upstream and in frame with the P2Apeptide. A 1084 bp fragment was cloned in as right homologous arm togenerate the MAP2-Nanoluc-KI donor vector. For the GFAP-Nanoluc-KI donorvector, a 1022 bp fragment right before the stop codon and a 1020 bpfragment after the stop codon was cloned into backbone vector as theleft and right homologous arm, respectively.

Example 3: Reporter iPSC Lines Generation

Prior to nucleofection, XCL1 iPS cells were maintained and passed usingAccutase (Life Tech., NJ) to make sure cells are growing in monolayer.On the day of nucleofection, single cell suspension cells were generatedusing Accutase followed by inactivation and washes with HBSS. 4-6 ug ofeach pair of TALENs/ZFNs RNA was used for nucleofection using AmaxaHuman Stem Cell Nucleofection Kit (Lonza, NJ). After nucleofection,cells were plated in mTeSR™1 medium with 10 uM Rock inhibitor. After 2-5days recovery, cells were treated with appropriate antibiotics.Specifically, 2.5 μg/ml Puromycin (Life Tech., NJ) for AAVS1-copGFP,MAP2-nanoluc-KI and GFAP-Nanoluc-KI lines and 500 μg/ml Neomycin (LifeTech., NJ) for Chr13-copGFP line. Drug resistant colonies were re-platedat low density for single cell cloning. Colonies growing from singlecells were screened by PCRs and sequencing to identify targets withcorrect donor vector integrations. The verified targets were expanded,stored and characterized for future experiments.

Example 4: NSC Derivation and Neural Differentiation

Generation of NSC was accomplished in accordance with proceduresdescribed by Swistowski et al. (PloS one 2009 4:e6233). Morespecifically, NSC were derived from iPSC lines and were cultured onMatrigel coated dishes in Neurobasal® medium supplemented with 1%nonessential amino acids, 1% GlutaMAX, 1×B-27®, and 10 ng/ml bFGF, andpassaged using Accutase. Neuronal differentiation was achieved byculturing NSC in Neuronal Primer media (Xcell inc, CA) on a surfacecoated with Poly-L-ornithine (2 μg/ml, Sigma, St. Louis, Mo.) andlaminin (10 μg/ml, Life Tech., NJ) at a density of 40-50 k/cm2 for 5-6days until cells become confluent. Then cells were split with Accutaseand were plated onto new poly-ornithine/laminin coated dishes at 40-50k/cm2 in Neuronal Medium (Xcell inc, CA) to continue differentiation foras long as desired. Astrocyte differentiation from NSC was also carriedout on culture dishes or glass cover slips coated withPoly-L-ornithine/laminin in Astrocyte Primer medium (Xcell Inc, CA).Medium was changed every other day and cells have to be split at least 3times before day 15. On day 18, change media to Astrocyte medium (Xcellinc, CA) and continue differentiation for up to day 35.

Example 5: Cre Recombinase-Mediated Cassette Exchange in iPSC and NSC

The iPSC or NSC master lines (AAVS1-copGFP or Chr13-copGFP) were platedon Matrigel-coated 35 mm dishes. When cells reached 70-80% confluency,plasmid expressing Cre recombinase and daughter construct wereco-transfected using Lipofectamine 3000 (Life Tech., NJ) followingmanufacturer's protocols. For iPSC, cells were selected with appropriateantibiotics to enrich the cell populations with successful cassetteexchange. Then drug resistant colonies were further screened using afluorescence microscope to identify colonies that lost greenfluorescence, which were picked, expanded and confirmed by PCR andsequencing.

Example 6: Immunocytochemistry

Immunocytochemistry and staining procedures were performed in accordancewith procedures described by Zeng et al. (Stem Cells 2003 21:647-653).Specifically, cells were fixed with 4% paraformaldehyde for 20 minutesat room temperature, blocked in blocking buffer (10% goat serum, 1% BSA,0.1% Triton X-100) for one hour followed by incubation with the primaryantibody at 4° C. overnight in 8% goat serum, 1% BSA, 0.1% Triton X-100.Appropriately coupled secondary antibodies, Alexa488 and Alexa594(Molecular Probes and Jackson ImmunoResearch Lab Inc.) were used forsingle or double labeling. All secondary antibodies were tested forcross reactivity and non-specific immune-reactivity. The followingprimary antibodies were used: Nestin (BD Biosciences, CA), GFAP(DakoCytomation Inc, CA), MAP2 (Sigma, St. Louis) and DCX (Santa CruzBiotechnology, TX). DAPI was used to label the nuclei.

Example 7: Luciferase Activity Measurement and HaloTag Detection

Determination of Nanoluc luciferase activity was measured using Nano-GloAssay System following manufacturer's protocol (Promega, WI).Specifically, 50 μl culture media was mixed with 50 μl of Nano-Glo AssayReagent in a 96-well plate for an incubation period of 5 min. Thenluciferase activity was measured using a Perkin Elmer Fusion-alpha-FP-HTuniversal microplate analyzer. Detection of HaloTag was achieved eitherin live cells using HaloTag® TMR Ligand following manufacturer'sprotocol (Promega, WI) or in fixed cells using HaloTag antibodies(Promega, WI).

What is claimed is:
 1. A master cell line integrated with a reportercassette at a safe harbor site in the cell, wherein said reportercassette comprises a reporter gene driven by a constitutively activepromoter and multiple Lox sites.
 2. The master cell line of claim 1wherein a Cre-recombinase induced cassette exchange strategy is used toexchange said reporter cassette in the master cell line with a newreporter cassette, thereby generating a new reporter line with the sameisogenic background.
 3. The master cell line of claim 1 wherein thepromoter driving the reporter gene is inserted between two Lox sites. 4.The master cell line of claim 1 wherein the promoter is inserted betweenlox2272 and lox511.
 5. The master cell line of claim 1 wherein the cellis a progenitor cell.
 6. The master cell line of claim 1 wherein thecell is a pluripotent or multipotent stem cell.
 7. The master cell lineof claim 6 wherein the cell is an induced pluripotent stem cell.
 8. Themaster cell line of claim 6 wherein the cell is a neural stem cell. 9.The master cell line of claim 1 wherein the cell is a differentiatedcell.
 10. The master cell line of claim 1 comprising a first reportercassette at a safe harbor site on chromosome 13 and a second reportercassette at a safe harbor site on chromosome 19, wherein said first andsecond reporter cassettes each comprise a reporter gene driven by aconstitutively active promoter and multiple Lox sites.
 11. A method ofgenerating a new reporter line, said method comprising utilizing aCre-recombinase induced cassette exchange strategy in the master cellline of claim 1 to exchange the reporter cassette in the master cellline with a new reporter cassette, thereby generating a new reporterline with the same isogenic background.